Maintenance of the adult male germline is essential for reproduction. Still a poorly understood process, germline stem cell self-renewal appears to be influenced by epigenetic chromatin modifications and subsequent nuclear organization. This proposal is designed to increase our understanding of these epigenetic regulatory processes. We hypothesize that histone modification and associated nuclear protein composition, including transcriptional repressor complexes and chromatin remodeling factors, are important determinants in whether stem cells self-renew or differentiate. Using mouse spermatogonia as a model, we will address the regulation of stem cell self-renewal in two specific aims. The first aim will examine the role of Polycomb group proteins on gene silencing in self-renewing spermatogonia. To achieve this aim, two distinct spermatogonial populations will be FACS-sorted and analyzed by transcriptional profiling. Potential Polycomb group binding sites will then be identified by chromatin immunoprecipitation (ChIP). Loss-of-function effects will be examined by one of two methods: generation of conditional knockout mice or RNAi knockdown and transplantation of cultured spermatogonial stem cells into recipient testes. The second aim will examine the role of nuclear matrix proteins in the maintenance of self-renewing spermatogonia. Proteomic profiling will be performed after fractionating nuclear matrix proteins from FACS-sorted germ cells and identifying their composition by liquid chromatography-mass spectrometry. Generation of conditional knockout mice or RNAi and transplantation experiments will then be performed to assess the functional importance of matrix-associated proteins. The proposed research for this award is designed to encompass both the mentored phase (K99) and the independent phase (R00), with a transition period built into the plan to successfully bridge the two phases. Relevance: A better understanding of how chromatin is modified and how these modifications influence gene expression during the self-renewal of stem cells will greatly contribute to the emerging field of regenerative medicine. Recent reports of pluripotent embryonic stem cell-like colonies emanating from cultured male germline stem cells warrant further investigation into the epigenetic control of stem cells in the testis. This proposal is both highly relevant and timely in this regard by addressing fundamental biological questions concerning the chromatin structure and gene expression in self-renewing spermatogonia and their differentiated progeny. Furthermore, deficiencies in histone modifications have been linked to infertility, while altered nuclear matrix proteins have been implicated in cancer. This proposal thus encompasses research highly relevant to the goals of the NIH.